SUMMARY Influenza is a major public health risk. Current seasonal influenza vaccines are effective in protecting against closely matched viruses in healthy adults. Because continuous genetic changes occur in influenza, there are major limitations to seasonal influenza vaccines including the need to produce new vaccines every season, uncertainty in selecting vaccin strains, and the inability to prevent novel influenza pandemics. A universal influenza vaccine will overcome these challenges. In our previous and preliminary studies, we have produced double-layered protein nanoclusters by desolvating the conserved ectodomain of the influenza M2 protein (M2e) into nanoparticles as cores and crosslinking influenza A trimeric hemagglutinin (HA) stalk antigens onto the core surfaces. We have also desolvated influenza internal nucleoprotein (NP) into particulate cores and cross linked M2e to generate double-layered nanoclusters. The resulting layered nanoclusters induced cross protection against viruses from both phylogenetic groups of influenza A, including pandemic potential avian strains. Both influenza A and B can cause epidemics. In this proposal, we will develop a multivalent double-layered nanocluster universal influenza vaccine composed of newly designed, conserved, antigenic proteins from both influenza A and B, and a molecular adjuvant. This nanocluster vaccine will induce strong cross immune protection against both influenza A and B in different laboratory animal models. The optimal nanocluster formulation will be encapsulated into dissolvable microneedle (MN) patches to develop a syringe-free, painless, thermostable, and self-administered skin-given universal influenza vaccine. The three specific aims are: Aim 1. Generate constructs of trimeric HA stalk antigen from influenza B and conserved NPs from both influenza A and B, fabricate and characterize nanoclusters from these and previously designed conserved antigenic proteins. We have generated structure-stabilized HA stalk proteins from both phylogenetic groups of influenza A (hrH1 and hrH3) and tetrameric M2e. We will fabricate novel double-layered nanoclusters from previous and new designed conserved influenza antigenic proteins. Aim 2. Test whether the layered nanoclusters or a multivalent optimal combination will induce protection against viruses spanning both influenza A and influenza B in mice. We will optimize a vaccine formulation inducing broadly reactive immune responses and cross protection in mice and further studies in Aim 3. Aim 3. Encapsulate the optimal multivalent nanocluster formulation into dissolvable MN patches and test the breadth of protection of the MN-based skin vaccination in both mice and ferrets. Dissolvable MN patch-based skin influenza vaccination has many advantages over conventional syringe injection including painless, needle-free, self-administration and cold chain-independent distribution. Overall, our research will develop a broadly cross-protective universal influenza vaccine